Seven-transmembrane receptors (7TMRs), also known as G protein-coupled receptors (GPCRs), serve cardinal roles in the regulation of cardiovascular function and are the targets of many of the most prevalent therapies for cardiovascular disease (e.g., the 2-adrenergic receptor (2AR) targeted by -blockers). Activation of these receptors has long been known to initiate signaling cascades through the action of G proteins. More recent discoveries have demonstrated that 7TMRs also initiate non-G protein-dependent signaling events mediated by -arrestins (arrs), which, in addition to their originally characterized role in 7TMR desensitization, serve as scaffolds for a variety of signalin proteins. It has further become apparent that certain ligands can preferentially activate subsets of 7TMRs' multiple downstream effects, a phenomenon known as biased agonism that could be exploited to develop biased drugs with fewer off-target effects. However, characterization of the molecular determinants of biased signaling and investigation of this phenomenon's therapeutic potential have been hindered by the dearth of robust reagents for probing bias. Accordingly, the objective of this proposal is to identify a series of biased mutants of the 2AR for use in diverse studies. The 2AR has been characterized in vivo and in vitro far more extensively than any other 7TMR, making it a logical target for studies of biased signaling, but no strongly biased 2AR ligands are known. Since receptor mutagenesis is a validated alternative to ligands for biasing 7TMR signaling, we aim first to systematically screen libraries of 2AR mutants for those exhibiting biased signaling. We will construct focused, semi-rationally designed libraries of mutants and blindly screen constructs for G protein activation, arr recruitment, and expression in a multiplexed, medium-throughput assay format. Second, we will subject candidate mutants from this screen to a panel of assays to confirm their bias more rigorously and evaluate their potential for use in downstream applications. Successful completion of these objectives will provide a valuable repository of tools for the study of biased signaling, which could ultimately guide the design of next- generation medications targeting the 2AR and other 7TMRs that possess novel therapeutic profiles and improved specificities compared to existing drugs.